Folia Morphol. Vol. 63, No. 1, pp. 1–3 Copyright © 2004 Via Medica R E V I E W A R T I C L E ISSN 0015–5659 www.fm.viamedica.pl Cell cycle checkpoints — molecular background Barbara Grzelakowska-Sztabert Nencki Institute of Experimental Biology, Warszawa, Poland [Received 11 September 2003; Accepted 30 December 2003] Cell cycle checkpoints are the surveillance mechanisms monitoring both the fi- delity and accuracy of DNA replication and the segregation of chromosomes. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication and ensure the proper segre- gation of chromosomes during mitosis. The paper provides basic information about the molecular mechanisms operating in various cell cycle checkpoints ac- tivated by DNA damage or disturbances in mitotic spindle assembly. key word: Cdk kinases, ATM, p53, DNA damage, mitotic spindle INTRODUCTION other events of the cell cycle progression. If the dam- The cell cycle consists of DNA synthesis (S) and age is irreparable, checkpoint signalling activates path- mitosis (M) phases separated by gap (G) phases in ways that lead to programmed cell death. Loss of the order G1-S-G2 M. It is driven by the sequential checkpoint integrity can allow the propagation of DNA activation of a number of Ser/Thr protein kinases that lesions and result in permanent genomic alterations. become active when they associate with their respec- Most of the information about molecular mechanisms tive regulatory cyclin subunits. Each of these cyclin- and proteins involved in cell cycle checkpoints has dependent kinases (Cdk kinases) phosphorylates and been obtained in genetic studies of budding and fis- therefore alters the proteins required for the execu- sion yeasts, in which mutants defective in particular tion of specific cell cycle events. The activities of Cdk phases of the cell cycle were produced. Nowadays kinases are regulated by changes in the supply of the we have already discovered that an essential mecha- cyclin subunit (proteins that exhibit the characteristic nism for the maintenance of genomic integrity exhib- pattern of appearance and disappearance during the its a high degree of evolutionary conservation in eu- cell cycle), by inhibitory or activatory phosphorylations karyotes and that multiple mechanisms could control of the catalytical subunit and by association with dif- the same cell cycle transition point. This enables the ferent inhibitory proteins (Cdk inhibitors) [1, 5]. Pro- checkpoint to respond to different types of stress and gression from one phase of the cell cycle to the apply precise control. next can be initiated only after passage through Two main classes of checkpoint can be distin- what are known as checkpoints (a concept intro- guished, one connected with cellular responses to duced by L. Hartwell, Nobel laureate in 2001 [7]), DNA damage and the other occurring during mitosis. where correct completion of the preceding steps is DNA damage-induced checkpoints verified. Checkpoints, therefore, may be considered as surveillance mechanisms controlling the order and Checkpoints activated by various types of DNA timing of cell cycle events and ensuring that biochem- damage may occur at the border of G1/S phases ically independent processes are completed. A delay (G1 phase checkpoint), during the S phase (intra-S in a critical cell cycle process will cause a delay in all phase checkpoint, DNA replication checkpoint, genome Address for correspondence: Barbara Grzelakowska-Sztabert, Nencki Institute of Experimental Biology, ul. Pasteura 3, 02–093 Warszawa, Poland, tel: +48 22 659 3072, fax: +48 22 822 53 22, e-mail: [email protected] 1 Folia Morphol., 2004, Vol. 63, No. 1 integrity checkpoint), during G2 and at the border interferes with its ability to bind p53, which con- of G2/M phases (G2 damage checkpoint, G2/M tributes to the stabilisation and nuclear accumula- phase checkpoint) [11]. These were originally de- tion of p53. fined as regulatory pathways that control the abil- The S-phase checkpoint is of particular impor- ity of cells to arrest the cell cycle in response to tance, since duplicaton of the genome takes place DNA damage, allowing time for repair. It is now in the S phase and repair of damaged DNA in this known that, in addition to controlling cell cycle ar- phase may be the final line of defence to eliminate rest, these pathways also control DNA repair path- DNA lesions before they are converted into inherit- ways, various transcriptional programmes and te- able mutations. A number of mechanisms have lomeres length as well as induce routes leading to been identified for the attenuation of the S-phase cell death by apoptosis. in response to DNA damage. The main mechanism Three major groups of proteins: sensors, transduc- includes sequential action of ATM and Chk2 kinas- ers and effectors are known which act in concert to es, which leads to inhibitory phosphorylation of translate the signal of damaged DNA into a cellular Cdc25A phosphatase, responsible for Tyr15 dephos- response. As sensor proteins that recognise damaged phorylation and therefore activation of all Cdk ki- DNA directly or indirectly and signal the presence nases. The other is the pathway ATM-Nbs1, which of abnormalities the following are proposed: probably participates in the inactivation and sub- poly(ADP-ribose) polymerase, DNA-dependent pro- sequent degradation of Cdc25C phosphatase. The tein kinase, as well as proteins related in structure other downstream factors of this pathway, proba- to PCNA (Rad 9, Rad 1, Hus 1) or subunits of repli- bly proteins involved in DNA replication, are yet to cation factor C (Rad 17). Among transducer pro- be identified. teins two protein kinases, ATM and ATR, related to In the G2 phase checkpoint, which controls the the phosphoinositide 3-kinase, play the central role transition from the G2 phase to mitosis, the phos- in the entire DNA damage response [8]. These am- phatase activity of Cdc25C is of special importance, plify the damage signal from the sensors by phos- as it activates Cdk1 kinase complexed with cyclin B phorylating other kinases (e.g. checkpoint kinases or A, indispensable for mitosis. Phosphorylation of Chk1 and Chk2) and other effector proteins involved Cdc25C phosphatase by Chk1 kinase, activated ei- in DNA repair, transcription regulation and cell cy- ther by ATM or ATR, provides an effective G2/M block cle control such as BRCA1, Nbs1(Nibrin), p53 and upon recognition of DNA damage. phosphatase Cdc25. Checkpoints during mitosis In the regulation of the G1 checkpoint the tumour suppressor protein p53 is the major damage effector During mitosis at least two checkpoints are protein [2]. It prevents the onset of the S-phase present. The first is named the CHFR checkpoint, af- through transcriptional regulation of p21, a Cdk ter a gene encoding a protein which possesses Fork- kinases inhibitor, as well as GADD45, a protein able head-associated and RING finger domains as well as to complex with PCNA, or 14-3-3, proteins that rec- ubiquitin ligase activity [3]. This occurs during ognise and bind proteins containing phosphorylat- prophase when CHFR protein inhibits chromatin con- ed serines. As a result, the activity of Cdk2 kinase densation and therefore entry into the metaphase. complexed with cyclin E, necessary for S-phase ini- The second checkpoint is situated between the tiation, is suppressed. In this situation suppressor metaphase and anaphase and is named the “spin- protein Rb remains unphosphorylated and bound dle assembly” checkpoint, mitotic checkpoint, or to transcription factor E2. Moreover, the transcrip- spindle checkpoint [10]. It monitors the microtubule tion of genes coding proteins required for DNA syn- structure and chromosome attachments to the mi- thesis is still blocked. An elevated intracellular level totic spindle and delays chromosome aggregation of p53 protein is observed in cells treated with ion- during the anaphase, until defects in the mitotic ising and UV irradiation, mainly as a result of its spindle apparatus are corrected. Crucial constituents increased stability. Phosphorylation of p53 by ATM of this checkpoint are the kinetochore-associated on Ser15 and by ATM-activated Chk2 kinase on proteins, designated as MAD2, BUBR1, BUB1 and Ser20 prevents its targeted degradation in protea- BUB2, which are able to form multicomponent com- some 26S. Additionally, phosphorylation by ATM plexes. These act in concert and regulate (especially of MDM2 cellular protein, acting as ubiquitin ligase, MAD2 and BUBR1) mitotic progression by direct in- 2 Barbara Grzelakowska-Sztabert, Cell cycle checkpoints teraction and inhibition of the APC ubiquitin ligase REFERENCES responsible for the proteolytic degradation of mul- 1. 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